INGLESE

English

In the first part of this course (NMR) the principles of how obtain and evaluate complex spectra
and structure elucidation are provided. Theoretical basis of bio-NMR spectroscopy are given
such as: the vector model, relaxation, nuclear Overhauser effect, scalar and dipolar coupling,
chemical shift. NMR signal assignment and multidimensional NMR spectroscopy is introduced.
Basics of NMR structure calculation will be described, including data collection, resonance
assignment, collection of structural restraints. The most advanced NMR techniques for studying
protein-protein and protein-ligand interactions will be illustrated such as: STD, transfer NOE,
WaterLOGSY, Chemical Shifts perturbation.
In the second part of this course (X-ray) the main theoretical and practical concepts for
structural characterization of biomolecules are given such as: preparing crystals, preliminary
characterization, data collection, solution of the phase problem, refinement and structure

Understanding NMR Spectroscopy, 2nd Edition.

Protein NMR Spectroscopy: Practical Techniques and Applications.

BioNMR in Drug Research (Methods and Principles in Medicinal Chemistry).

Fundamentals of Crystallography I- International Union of Crysyallography.

Practical Crystallography - Pergamon Press.

The aim of this course is to introduce the theory and practice for the structural characterization
of biomolecules by using NMR spectroscopy and X-ray diffraction techniques, including the
required data acquisition, processing steps and required computer software. At the end of the
course the student will learn how to obtain NMR and X-ray data to characterize the structural
proprieties of biomolecules. Moreover, the student will learn the most advanced NMR
methodologies for studying Protein-protein and Protein-ligand interactions. During the course
the student will be involved in several training activities on the spectrometers located in the
NMR laboratory of the Department of Environmental, Biological and Pharmaceutical Science and
Technologies in order to fully understand the procedure for NMR data acquisition.

No propedeutics
Basic knowledge of Inorganic and Organic Chemistry, Mathematics, Physics, Biochemistry and Biology.

The attendance to the lessons is mandatory. The course is organized in 52 hours of frontal lessons that will be theoretical and practical. Further 12 hours will be dedicated to the lab practice ( six 2 hours practices). During such experiences, that will be performed in groups of two or three components, the students will learn how to characterize the structural the molecular and supramolecular structure of Biomolecules by using NMR and X-ray data. Moreover, During the course
the student will be involved in several training activities on the spectrometers located in the
NMR laboratory of the Department of Environmental, Biological and Pharmaceutical Science and
Technologies in order to fully understand the procedure for NMR data acquisition.

The final examination will consist in an oral interview, aimed to verify the notion acquired and the ability of the student to re-elaborates the contents presented during the course. In particular, the oral examinations are aimed to evaluate the capability of reasoning and connecting the various arguments of the course and, i.e. the basic concepts of NMR, the application of NMR to drug discovery and the basic concepts of X-ray crystallography, is constituted by questions about the theoretical part of the course to evaluate the knowledge of the studied subjects and the capability to organize the exposition and connection of the diverse arguments.

During the course slides and notes of the lectures will be provided to the students via the Web site of the University of Campania “L. Vanvitelli” (sharepoint)
Each Professor is available for student reception during the office hour as reported in the course sheet or by e-mail request.

Fundamentals of modern NMR spectroscopy: Spin magnetization; Vector model; Rotating
frame of reference; Pulses (Soft and Hard); Fourier transformation; Spectral processing; NMR
parameters: Chemical Shifts, J scalar couplings; integration; Nuclear Overhauser Effect (NOE);
Dipolar coupling; Concepts of the Multidimensional NMR experiments; Analysis of pulse
sequences 1D and 2D experiments (homo- and hetero-nuclear): 1D 1H; 2D TOCSY; 2D NOESY,2D
ROESY, 2D COSY;2D 1H-15N HSQC; 1H-13C HSQC; Spin-lock; correlation time; constant time;
Relaxation theory: Correlation function; Longitudinal Relaxation rate (R1); Transverse Relaxation
rate(R2); Mechanisms inducing relaxation: Chemical Shifts Anisotropy; Dipolar Coupling;
Experiments for measuring relaxation parameters: Inversion recovery (2D 1H-15N HSQC) R1,
Spin-echo (2D 1H-15N HSQC) R2, heteronuclear [1H]-15N NOE. Parts of the NMR spectrometer;
the superconducting electromagnet, the sample probe, the pneumatic unit, the preamplifier,
the console, the computer. Introduction of the basic operations for acquiring NMR spectra:
loading the sample, locking, tuning and shimming, setting acquisition and processing
parameters.
Biomolecular NMR spectroscopy: Use of NMR for biomolecular structure elucidation;
introduction of 3D triple resonance experiments; spectral assignments experiments: examples
HNCO; CBCA(CO)HN; CBCANH; HNCA; Overhauser effect Example 13C/15N-resolved NOESY;
software for processing and analysis of spectra: SPARKY, CARA, NMRDRAW; protocols and
software for structure calculation: Cyana, TALOS, PALES; Analysis and validation of the
calculated structures: PROCHECK; PSVS and PROCESS server
Residual dipolar coupling measurements Example: IPAP-HSQC; aligned media (polyacrylamide
gel (Compressed and Stretched), Pf1 Phage); Principles of TROSY Spectroscopy; Relaxation
measurements, Models of motions, Protein dynamics: Example 15N T1; 15N T2; T1ρ; Het [1H]-
15N NOE.
NMR in Drug Discovery: Physical effects of protein-ligand interactions relevant for NMR:
thermodynamics of ligand binding; kinetics of ligand binding; Exchange regime (Fast,
Intermediate, slow); chemical environment and interactions; Brownian motion (rotational and
translational diffusion); NMR observable parameters associated with basic physical effects; How
ligand binding effects influence the NMR signal; Protein observed chemical shift titrations:
Chemical Shifts Mapping Example Heteronuclear HSQC detected titrations; Ligand observed
methods: Ligand observed relaxation rates, chemical shifts, transverse and longitudinal rates,
translational diffusion, magnetization transfer (Saturation Transfer Difference NMR STD,
WaterLOGSY, transfer NOE).Example of Protein-ligand studies observing Protein or Ligand
Biocrystallography: Introduction, role of biocrystallography in macromolecular structural
chemistry; Properties of crystals. Symmetry; Symmetry elements. Space groups; Reciprocal
lattice; Unitary Cell - Miller Indices; Classification of crystalline lattices; - The 14 lattices of
Bravais; X-ray diffraction; The Bragg Law; Diffraction Conditions; Structural Factors; X-ray
sources; X-ray generators; Automatic diffractometers; Data collections; Crystallization of
Macromolecules; Crystallization techniques of small and large molecules; crystal quality
evaluation; use of polarized microscope; The diffraction data; Determination of the elementary
cell; Calculation of the density of a crystal and of the number of molecules contained in the unit
cell of a macromolecular crystal; Diffracted intensity; Scattering factor; Resolution and
refinement of crystallographic structures; Problem of the phase; Data collection methods;
Solving the phase problem; The method of multiple Molecular replacement; Other methods
used to solve the problem of the phase: "trial and error" methods; "direct methods"; methods
of Patterson; Crystallographic refinement; Index of disagreement R.